This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The multiple-stage ion-trap mass spectrometric approaches towards to the structural characterization of the monoacyl-PIM (triacylated PIM) and the diacyl-PIM (tetracylated PIM), namely, the PIM (diacylated PIM) consisting of one or two additional fatty acid substituents attached to the glycoside, respectively, were described. While the assignment and confirmation of the fatty acid substituents on the glycerol backbone can be easily achieved by the methods described in the previous article, the identity of the glycoside moiety and its acylation state can be determined by the observation of a prominent acylglycoside ion arising from cleavage of the diacylglycerol moiety ([M - H - diacylglycerol]-) in the MS2 spectra of monoacyl-PIM and diacyl-PIM. The distinction of the fatty acid substituents on the 2-O-mannoside (i.e, R3CO2H) from that on the inositol (i.e., R4CO2H) is based on the findings that the MS3 spectrum of [M - H - diacylglycerol]- contains a prominent ion arising from further loss of the fatty acid at the 2-O-mannoside (i.e, the [M - H - diacylglycerol - R3CO2H]- ion);while the ion arising from loss of the fatty acid substituent at the inositol (i.e, the [M - H - diacylglycerol - R4CO2H]- ion) is of low abundance. The fatty acyl moiety on the inositol can also be identified by the product-ion spectrum from MS4 of the [M - H - diacylglycerol R3CO2H]- ion, which gives rise to a prominent ion corresponding to loss of R4CO2H. An [M - H - acylmannose]- ion was also observed in the MS2 spectra and thus, the identity of the fatty acid substituent attached to 2-O-mannoside can be confirmed. The combined information obtained from the multiple-stage product-ion spectra from MS2, MS3, and MS4 permit the assignment of the complex structures of monoacyl-PIMs and diacyl-PIMs in a mixture isolated from M. bovis Bacillus Calmette Guerin.